We believe that eradication of malaria is achievable but not without a potent vaccine that interrupt malaria transmission (VIMT) transmission blocking vaccine. A logical and promising strategy is to combine target antigens from multiple stages to potently prevent transmission. To prevent transmission a vaccine should target the pre-erythrocytic (sporozoite and liver stages) and the sexual-mosquito stages of the life cycle. An ideal malaria vaccine would prevent infection, disease, and transmission by targeting at a minimum the pre- erythrocytic (sporozoites and liver stages) and optimally other stages of the parasite life cycle also. Pre- erythrocytic stage vaccine development is based on the observation that immunization via bites of irradiated mosquitoes infected with Plasmodium falciparum (Pf) sporozoites (SPZ) provides high-level protection. The circumsporozoite protein (CSP) was discovered by immunizing mice with irradiated SPZ. A higher percent of volunteers immunized with radiation attenuated PfSPZ have T cells that recognize Pf cell-traversal protein for ookinetes and sporozoites (PfCelTOS) than PfCSP, and immunization of mice with PfCelTOS protects against challenge with rodent malaria SPZ at the pre-erythrocytic stage. We have discovered that antibodies induced in mice by immunizing with recombinant (r) PfCelTOS with adjuvant inhibited Pf development to oocysts in mosquitoes in vivo and PfSPZ invasion and development in hepatocytes in vitro. When mice were immunized with rPfCelTOS alone, rPfCSP alone, or both, mice immunized with both proteins had higher Abs against PfSPZ and activity in blocking SPZ invasion and development in hepatocytes (86%) than did mice immunized with either protein individually. The observations that antibodies against rPfCelTOS, had biological activity against parasite mosquito (ookinete) and pre-erythrocytic (SPZ) stages, and were additive or synergistic with anti-PfCSP antibodies against SPZ are unique, and argue for further development of this protein. To further enhance VIMT effects, we will assess Pf von Willebrand factor A domain-related protein (PfWARP), a highly conserved, soluble ookinete specific protein that we have shown previously to potently inhibit development of oocysts in the mosquito midgut. Our aim in this study is to develop a combined multiple stage vaccine to potently prevent transmission by inhibition of oocyst development. PfCelTOS and PfCSP are also expressed in hemocoel stage sporozoites and thus our strategy would also target the conversion of oocysts to infectious sporozoites in the salivary glands. We will study the three proteins as immunogens alone and in combination, aiming to induce 100% transmission blocking activity. We believe that eradication of malaria is achievable but not without a potent transmission blocking vaccine, and that these 3 proteins can achieve this goal. Such a vaccine would be used in infants, young children, adolescent females (prevent malaria in pregnancy) and malaria elimination campaigns as a public health measure; an enormous global health market. PUBLIC HEALTH RELEVANCE: Malaria causes 400-500 million clinical cases and >1 million deaths annually, is responsible for >1% loss of GDP in Africa annually and is a serious concern for travelers and military personnel. We believe that eradication of malaria is achievable but not without a potent transmission blocking vaccine. A logical and promising strategy is to combine multiple stage targets to potently prevent transmission. Such a vaccine would have huge public-sector markets. In public sector markets it would be used in infants, young children, adolescent females (prevent malaria in pregnancy) and malaria elimination campaigns as a public health measure.